Cell division is ordinarily controlled in multicellular organisms such that cells grow within certain bounds, at certain rates, and adjacent to certain other cells. Cells derived from such organisms exhibit such controlled growth even when placed in culture. Thus, for example, these cells will grow in a petri dish until a certain density of cells occupies the dish, thereafter attaining a quiescent, non-dividing or very slow dividing stage. This characteristic of normal cells is a function of some combination of cellular contact and the concentration of a factor or factors that accumulates in the medium in which the cells are plated. Other properties of normal cells include: oriented growth, lack of fetal or virus-specific antigens on the cell surface, high serum requirement for growth, inability to grow in agar, and inability to cause tumor formation upon injection into susceptible animals.
However, within any given culture, and within any given multicellular organism, a proportion of the cells will lose their characteristic of controlled growth, and proliferate to excess. Such cells are referred to as "transformed," which cells may grow in culture to saturation densities that are 10-25 times greater than those of nontransformed normal cells, when nutritional factors probably become limiting. Transformation can be induced using any of various agents, including certain viruses and chemicals. Transformation may also be an inherent property of normal cells, such that at some rate, all cells will transform. Transformed cells include tumor or cancer cells, whether in vivo or in vitro, such as cells from non-small cell lung cancer, small cell lung cancer, colon cancer, melanoma, ovarian cancer, and renal cancer.
Agents that specifically, preferentially, or predominantly inhibit or stop the growth of transformed cells have utility for the negative selection of transformed cells as well as for use in chemotherapy for various forms of cancer. Thus, whether in vivo or in vitro, transformed cells exhibiting uncontrolled growth would be inhibited or killed by such agents, while allowing the generally slower growing nontransformed, normal cells to survive unfettered (or less fettered) by the transformed cells. Such agents generically are classified as cytotoxins, among which are alkylating agents.
The cytotoxic agent 2,9-dimethyl-1,10-phenanthroline ("2,9-DMP") is a potent, copper dependent cytotoxin, whose administration as a chemotherapeutic agent is limited by its neurotoxic characteristic. The coordinated copper complex of 2,9-DMP has been shown to be effective in tissue culture at nanomolar concentrations for the killing of mouse L1210 cells, for example, which is an established murine lymphoma line. When L1210 cells were implanted into a susceptible animal, and a tumor began growing therefrom, the observed inhibitory responses were evident, although weak, when the L1210-implanted animals received 2,9-DMP with copper. Mice inoculated with P388 murine lymphocytic leukemia cells, however, responded well to such 2,9-DMP treatment, resulting in 45% increases in life span in the treated group. Despite the positive result with the P388 cells, however, 2,9-DMP is not considered a good candidate for widespread use because it is a known neurotoxin. See Mohindru et al., Biochem. Pharmacol., 32, 3627-3632 (1983).
The parent compound to 2,9-DMP is 1,10-phenanthroline ("phenanthroline"), which also has cytotoxic capabilities. Phenanthroline has known antibacterial, antifungal, antiviral, and antineoplastic characteristics. See Blank, Nature, 168, 516-517 (1951); Butler et al., Aust. J. Exp. Biol., 47, 541 (1969); Shulman et al., Arzneimittel-Forsch, 22, 154 (1972); Shulman et al., Chem. Biol. Interact., 6 407 (1973); Shulman et al., Chem. Biol. Interact., 16, 89 (1977). Its cytotoxic characteristic is believed to be a function of its ability to chelate various divalent metal ions, such as zinc, which would thereby inhibit, for example, zinc-dependent nucleotidyl transferases and DNA synthesis. In each of the systems studied whereby the various cytotoxic effects have been observed, the addition of divalent metal ions has reversed the toxic effect. See Berger et al., Exp. Cell Res., 96, 145 (1975); Falchuk et al., Cancer Res., 37, 2050 (1977). However, various alkylated derivatives of phenanthroline complexed with any of the divalent metal ions Cu.sup.++, Co.sup.++, Fe.sup.++, Zn.sup.++, and Ru.sup.++ have been reported to be cytotoxic, albeit the degree of toxicity was seen to vary from 100% kill down to only 6% kill in 6 hours incubation (Shulman 1977, supra). In none of the studies, thus far, has a cytotoxic alkylating compound been identified that selectively inhibits transformed cells while leaving normal ones unaffected or affected in a limited or minor way.
There remains a need, therefore, for a compound that allows for the selection of nontransformed cells by inhibiting the growth of transformed cells. Such a compound preferably would be toxic to transformed cells while having little or no toxic effects on nontransformed cells. The present invention provides a compound and its method of use, which method allows for such a selection of nontransformed cells. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.